Extracellular nucleic acids have been identified in plasma, serum and other body fluids. Extracellular nucleic acids that are found in respective samples are to a certain extent degradation resistant due to the fact that they are protected from nucleases (e.g. because they are secreted in form of a proteolipid complex, are associated with proteins or are contained in vesicles). The presence of elevated levels of extracellular nucleic acids such as DNA and/or RNA in many medical conditions, malignancies, and infectious processes is of interest inter alia for screening, diagnosis, prognosis, surveillance for disease progression, for identifying potential therapeutic targets, and for monitoring treatment response. Additionally, elevated fetal DNA/RNA in maternal blood is being used to determine e.g. gender identity, assess chromosomal abnormalities, and monitor pregnancy-associated complications. Besides mammalian extracellular nucleic acids that derive e.g. from tumor cells or the fetus, samples comprising extracellular nucleic acids may also comprise other nucleic acids of interest that are not comprised in cells. An important, non-limiting example are pathogen nucleic acids such as viral nucleic acids. The efficient isolation of viral nucleic acids from samples such as in particular blood samples or samples derived from blood is also important for many diagnostic applications. Thus, extracellular nucleic acids are in particular useful in non-invasive diagnosis and prognosis and can be used e.g. as diagnostic markers in many fields of application, such as non-invasive prenatal genetic testing, oncology, transplantation medicine or many other diseases and, hence, are of diagnostic relevance (e.g. fetal- or tumor-derived nucleic acids). However, extracellular nucleic acids are also found in healthy human beings. Common applications and analysis methods of extracellular nucleic acids are e.g. described in WO97/035589, WO97/34015, Swarup et al, FEBS Letters 581 (2007) 795-799, Fleischhacker Ann. N.Y. Acad. Sci. 1075: 40-49 (2006), Fleischhacker and Schmidt, Biochmica et Biophysica Acta 1775 (2007) 191-232, Hromadnikova et al (2006) DNA and Cell biology, Volume 25, Number 11 pp 635-640; Fan et al (2010) Clinical Chemistry 56:8.
Thus, the efficient isolation of extracellular nucleic acids is of great importance in order to allow a reliable analysis. However, extracellular nucleic acids are often only comprised in a low concentration in the samples. E.g. free circulating nucleic acids are present in plasma in a concentration of 1-100 ng/ml plasma. Furthermore, extracellular nucleic acids often circulate as fragments of a size of 500 nt, 300 nt (when indicating the size and hence the chain length the term “nt” also includes “bp” in case of DNA) or even less (circulating nucleosomes). Additionally, the actual target extracellular nucleic acid that is supposed to be identified for diagnostic purposes usually also represents only a small fraction among the total extracellular nucleic acids. E.g. tumor specific DNA fragments are very rare and often are comprised in a concentration that is 1000-fold less than the “normal” extracellular nucleic acid background. Thus, it is desirous to process large sample volumes in order to obtain sufficient amounts of extracellular nucleic acids and in particular sufficient amounts of the rare target molecules contained therein for the downstream assays.
Several methods are known in the prior art for isolating extracellular nucleic acids from samples, such as in particular plasma samples. Here, also several kits are commercially available. However, even though these kits provide useful results, they have drawbacks which need improvement.
For example, the QIAamp circulating nucleic acid kit allows to process a sample size of up to 5 ml for isolating the extracellular nucleic acids. However, it requires a manual nucleic acids extraction. Automating a respective large volume sample preparation is difficult to implement because the respective kit needs to handle an overall process volume of up to 25 ml due to the used chemistry (lysis and binding buffers). Standard robotic systems can, however, only process a volume of up to 3 ml. Furthermore, the respective kit also requires several method steps. Therefore, a method would be advantageous which also allows the processing of large sample volumes but at the same time allows to automate the isolation process. Other commercially available kits which aim at the isolation of for example viral nucleic acids from cell-free samples such as plasma, can only process rather small sample volumes (see for example the ChargeSwitch® EasyPlex™ viral kit). Here, the processing of larger sample volumes would be advantageous, because this would allow to increase the nucleic acid yield.
Hence, the prior art methods have several drawbacks. To overcome the drawbacks of the prior art, it is desirous to provide a method for isolating extracellular nucleic acids from large sample volumes. This would ensure that sufficient extracellular nucleic acids are isolated for the downstream applications. If this requirement is not met, there is a risk that e.g. even sensitive methods are not capable of detecting the target molecules contained in the isolated extracellular nucleic acid population. Furthermore, the prior art methods are often time-consuming and thus require several handling steps and thus hands-on-time. Here, it is desirous to provide a simple, rapid method that requires only a few steps for isolating the extracellular nucleic acids. This would also reduce potential errors due to mistakes in the handling during the preparation. Furthermore, it is desirous to provide a method that is suitable for automation. Once a diagnostic target has been established for routine testing, customers require automation to manage higher throughputs e.g. in laboratories. An automated isolation protocol would have significant advantages in the diagnostic field because it would reduce the risks of erroneous results due to errors that occur during the manual nucleic acid isolation. However, robots which are designed to perform such automated nucleic acid isolation processes are limited by the maximum sample volume which they can handle (see above). Increasing the volume by addition of reagents necessary for performing the isolation process therefore directly reduces the amount of sample that can be processed and hence the amount of nucleic acid which can be obtained by a single run of the automated isolation process. Finally, the prior art kits usually require steps for lysing the sample. Respective lysis steps—which are commonly performed in the prior art—are not only required to e.g. release the nucleic acids from the cells. Respective lysis steps are also usually performed when isolation nucleic acids from so-called cell free samples such as plasma in order to denature and/or digest protein contaminations or other contaminating substances that could interfere e.g. with the binding of the nucleic acid to the solid phase and/or could lead to an improper purification. For performing the respective lysis step, often large volumes of lysis reagents such as e.g. chaotropic agents are added. The necessity to perform a respective volume increasing lysis step is a drawback, because the volume of the actual sample that can be processed e.g. in an automated system is reduced.
US 2005/0106602 describes methods of isolating nucleic acids from samples of cellular material. No chemical lysis is performed. Instead, nucleic acid binding groups are used, which serve a dual purpose, namely to bind the nucleic acids and to support the lysis of the cells. WO2006/036243 describes a similar method.
Melkonyan et al. Transrenal nucleic acids: “From proof of principle to clinical tests”, 2008, describes the isolation of extracellular nucleic acids from urine. The sample is contacted with a Q-Sepharose anion exchange matrix to bind the nucleic acids which are afterwards washed and eluted. The details of the used isolation protocol is described in Shekhtman et al. “Optimization of transrenal DNA analysis: Detection of fetal DNA in maternal urine” (Clinical Chemistry 55:4 page 723-729 (2009). Shekhtman et al. teaches to massively dilute the urine prior to binding the nucleic acids to the solid phase. 10 ml urine is diluted with 10 ml water and the resulting diluted sample is contacted with the Q-Sepharose. A similar method is described in WO2008/45505 which also describes a column based method for isolating cell-free nucleic acids from urine or blood plasma.
WO02/48164 describes a method of isolating nucleic acids using an anion exchange matrix and a charge-switch-procedure. At a first pH, the sample is brought into contact with a material which comprises an ionisable group, wherein the material has a positive charge at its first pH, such that nucleic acids are bound in the material. The nucleic acids are released at a second, higher pH at which the charge on the material is negative, neutral or less positive. The isolation of circulating nucleic acids, such as tumor derived extra cellular nucleic acid is not described.
DE 10 2008 063 003 describes a method for isolating nucleic acids using anion exchange surfaces. The isolation of extracellular nucleic acids using a large sample volume is not described. DE 10 2008 063 001 also discloses a method for isolating nucleic acids using anion exchange groups and specifically designed solid phases for binding nucleic acids.
Kirsch et al.: An improved method for the isolation of free-circulating plasma DNA and cell-free DNA from other body fluids, 2008, describes a method for isolating cell-free nucleic acids by using the NucleoSpin Plasma XS Kit (Macherey-Nagel). No anion exchange surface is used for binding the nucleic acids.
It is the object of the present invention to provide a method for isolating extracellular nucleic acids from a sample containing extracellular nucleic acids, which avoids at least one of the prior art drawbacks discussed above. In a specific embodiment it is an object of the present invention, to provide a simple, rapid method for isolating extracellular nucleic acids which is suitable for automation and allows to process large sample volumes.